New aspects of the blood-brain barrier transporters; its physiological roles in the central nervous system

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain, and restricts drug permeability into the brain. Our latest studies have revealed that the BBB transporters play important physiological roles in maintaining the brain milieu. The BBB supplies creatine to the brain for an energy-storing system, and creatine transporter localized at the brain capillary endothelial cells (BCECs) is involved in BBB creatine transport. The BBB is involved in the brain-to-blood efflux transport of the suppressive neurotransmitter, gamma-aminobutyric acid, and GAT2/BGT-1 mediates this transport process. BCECs also express serotonin and norepinephrine transporters. Organic anion transporter 3 (OAT3) and ASCT2 are localized at the abluminal membrane of the BCECs. OAT3 is involved in the brain-to-blood efflux of a dopamine metabolite, a uremic toxin and thiopurine nucleobase analogs. ASCT2 plays a role in L-isomer-selective aspartic acid efflux transport at the BBB. Dehydroepiandrosterone sulfate and small neutral amino acids undergo brain-to-blood efflux transport mediated by organic anion transporting polypeptide 2 and ATA2, respectively. The BBB transporters are regulated by various factors, ATA2 by osmolarity, taurine transporter by TNF-alpha, and L-cystine/L-glutamic acid exchange transporter by oxidative stress. Clarifying the physiological roles of BBB transport systems should give us important information allowing the development of better CNS drugs and improving our understanding of the relationship between CNS disorders and BBB function.     

Physiological function of blood-brain barrier transporters as the CNS supporting and protecting system

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain and restricts drug permeability into the brain. Our latest studies have revealed that the BBB transporters play important physiological roles in maintaining the brain environment. For an energy-storing system, the creatine transporter localized at the brain capillary endothelial cells (BCECs) mediates the supply of creatine from the blood to the brain. The BBB is involved in the brain-to-blood efflux transport of gamma-aminobutyric acid, and GAT2/BGT-1 mediates this transport process. BCECs also express serotonin and norepinephrine transporters. Organic anion transporter 3 (OAT3) and ASCT2 are localized at the abluminal membrane of the BCECs. OAT3 is involved in the brain-to-blood efflux of a dopamine metabolite, a uremic toxin, and thiopurine nucleobase analogues. ASCT2 plays a role in L-isomer-selective aspartic acid efflux transport at the BBB. Dehydroepiandrosterone sulfate and small neutral amino acids undergo brain-to-blood efflux transport mediated by organic anion transporting polypeptide 2 and ATA2, respectively. The BBB transporters are regulated by various factors: ATA2 by osmolarity, taurine transporter by tumor necrosis factor-alpha, and L-cystine/L-glutamic acid exchange transporter by oxidative stress. Clarifying the physiological roles of BBB transport systems should give important information allowing the development of better central nervous system (CNS) drugs and improving our understanding of the relationship between CNS disorders and BBB function.     

Blood-brain barrier transport of synthetic adenosine A1 receptor agonists in vitro: structure transport relationships.

Transport of 11 structurally related adenosine A(1) receptor agonists was determined in an in vitro BBB model of brain-capillary-endothelial-cells and astrocytes. Inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI) was used to quantify the contribution of the es nucleoside transporter to the overall transport. The N(6)-substituted adenosine analogues N(6)-cyclobutyladenosine (CBA), N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA) showed concentration-dependent clearance and their transport could be inhibited by NBTI. The V(max) was 1.5+/-0.2 pmol min(-1) and the Km values were 2.2+/-0.2, 1.8+/-0.3 and 15+/-4 microM for CBA, CPA and CHA, respectively. Further chemical modification such as substitution in the C8-position or modification at the ribose-moiety resulted in loss of affinity for the es nucleoside transporter. Transport by passive diffusion was slow with clearances ranging from 0.21+/-0.01 microl min(-1) for 8-(methylamino)-CPA (MCPA) to 1.8+/-0.18 microl min(-1) for 5'-deoxy-CPA (5'dCPA). Regression analysis showed no relationship between transport clearance by passive diffusion and the GTP-shift, a non-linear relationship between the transport clearance by passive diffusion and the dynamic polar surface area (Cl=0.469e(-0.071DPSA); R2=0.88) and a linear relationship between transport clearance and prediction of BBB transport on basis of the Abraham equation (logCl=1.53logBB-1.56; R2=0.83). It is concluded that the transport of synthetic A(1) adenosine derivatives across the blood-brain barrier is generally quite slow. In addition, transport by the es nucleoside transporter may contribute to the transport of certain structurally distinct analogues.     

Interaction of a series of draflazine analogues with equilibrative nucleoside transporters: species differences and transporter subtype selectivity

The equilibrative nucleoside transporters of mammalian cells play an important role in the regulation of extracellular adenosine concentrations, and inhibition of these transporters potentiates the biological effects of adenosine. Two subtypes of equilibrative transporters have been defined by their differential sensitivities to inhibition by nitrobenzylthioinosine (NBMPR; es/ENT1, sensitive; ei/ENT2, insensitive). In addition, significant species differences have been noted in es/ENT1 transporter affinity for a subset of inhibitors including draflazine and dipyridamole. Draflazine and a series of 15 chemically related compounds were compared for their abilities to: (a) inhibit the binding of [3H]NBMPR to the es/ENT1 transporter in mouse Ehrlich cell and human erythrocyte membranes, and (b) inhibit the es/ENT1 and ei/ENT2 transporter-mediated uptake of [3H]uridine in Ehrlich cells. Compounds within this series represented over a 1000-fold range of affinities for the es/ENT1 and ei/ENT2 transporters with subtype selectivities (ENT1/ENT2) ranging from 370 for R70527 to 0.17 for soluflazine. Five other analogues were identified, in addition to soluflazine, that had significantly higher affinity for the ei/ENT2 transporter compared with es/ENT1. Structure activity analyses of these data identified the requirement of a hydrophobic group connected to a 2-aminocarbonyl piperazine by a 5-carbon chain for high-affinity interactions with es/ENT1. This hydrophobic moiety was not as important for ei/ENT2 affinity and, in contrast to es/ENT1, a shorter alkyl chain enhanced binding to ei/ENT2. These draflazine analogues also varied in their differential affinities for mouse vs. human es/ENT1 transporters, and the degree of species discrimination was strongly dependent on the position of the aminocarbonyl group on the piperazine ring. This information, combined with structural data derived from molecular studies with ENT1 and ENT2 recombinant proteins, should guide further development of subtype-selective inhibitors of the equilibrative nucleoside transporters.     

Functional characterization of adenosine transport across the BBB in mice

We investigated transport characteristics of adenosine across the blood–brain barrier (BBB) in mice. Uptake clearance across the BBB was measured by using an in situ mouse brain perfusion technique and cultured mouse brain capillary endothelial cell line (MBEC4 cells). Nucleoside transporter was cloned by RT-PCR and expressed on Xenopus laevis oocyte. Both in situ and in vitro studies revealed that the adenosine uptake is concentration-dependent, Na⁺-independent and S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive. The K_t values of in situ and in vitro studies were 31.7 ± 13.8 μM and 11.9 ± 2.84 μM, respectively. A good correlation was found for the inhibitory effects of nucleoside analogs to adenosine uptake between in situ and in vitro studies. RT-PCR revealed the expression of RNA of mouse equilibrative nucleoside transporter (mENT1) in mouse brain capillary and MBEC4 cells. In mENT1 expressed on X. laevis oocyte, K_t value of adenosine transport was 6.9 ± 2.7 μM (and comparable to those in situ and in vitro studies). In conclusion, we characterized the adenosine transport across the BBB in mice by using in situ brain perfusion technique and MBEC4 cells and found that these transports share common characteristics with mENT1-mediated transport. Transport of adenosine across the BBB in mice may be attributable to mENT1.     

Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors.

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.     

Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes.

OBJECTIVE: The aim was to establish a flexible, abundantly available, reproducible and functionally characterized in vitro model of the blood-brain barrier (BBB). METHODS: In a first step, bovine brain capillaries and newborn rat astrocytes were isolated. Subsequently, a co-culture of primary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, with astrocytes on the bottom of the filter was established. The cell material was characterized on the basis of specific cell-type properties and (functional expression of) specific BBB properties. RESULTS: BCEC displayed: (1) characteristic endothelial cell morphology; (2) expression of endothelial cell markers (i.e., CD51, CD62P, CD71 and cadherin 5); (3) marginal F-actin localization; (4) tight junction formation between the cells; (5) expression of gamma-glutamyl-transpeptidase (gamma-GTP); (6) expression of P-glycoprotein (Pgp); (7) functional transendothelial transferrin transport and uptake; (8) restriction of paracellular transport; and (9) high transendothelial electrical resistance (TEER). Astrocytes displayed characteristic astrocyte morphology and expressed glial fibrillary acidic protein (GFAP). Co-culture with astrocytes increased TEER and decreased paracellular transport. In addition, expression of the glucocorticoid receptor (GR) was demonstrated in the endothelial cells of the BBB, while no expression of the mineralocorticoid receptor (MR) was found. CONCLUSIONS: A high quality and mass-production in vitro BBB model was established in which experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and pharmacodynamics) and pathophysiological (e.g., disease influence on BBB permeability) objectives can be reproducibly performed.     

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 microM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (1AA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 microM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 microM). Treatment of cells with UTP (100 microM), histamine (100 microM), or phorbol-12-myristate-13-acetate (PMA, 10 microM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.     

Evaluation of blood-brain barrier transporters by co-culture of brain capillary endothelial cells with astrocytes

To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as gamma-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [(3)H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [(14)C]benzoic acid was saturable, and the K(m) value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [(14)C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [(3)H]dehydroepiandrosterone sulfate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.     

Transport of a GABAA receptor modulator and its derivatives from Valeriana officinalis L. s. l. across an in vitro cell culture model of the blood-brain barrier

The roots and rhizome of Valeriana officinalis L . s. l. are therapeutically used for their sedative and sleep-enhancing effects. Some of the active compounds found in commonly used extracts are the sesquiterpenic acids, especially valerenic acid, which was recently identified as a GABA (A) receptor modulator. To interact with this receptor in the brain, substances such as valerenic acid and its derivatives acetoxyvalerenic acid and hydroxyvalerenic acid have to cross the blood-brain barrier (BBB). The aim of our study was to obtain BBB permeability data of these compounds for the first time and to elucidate possible transport pathways across our BBB in vitro model. Transport of valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid was compared with the permeability of the GABA (A) modulator diazepam, which is known to penetrate into the central nervous system transcellularly by passive diffusion. Experiments were carried out with an established Transwell in vitro model based on the human cell line ECV304. Results indicated clearly that all three acids permeated significantly slower than diazepam. The ranking was confirmed in group studies as well as in single-substance studies after normalization to diazepam. Valerenic acid (1.06 +/- 0.29 microm/min, factor 0.03 related to diazepam) was the slowest to permeate in the group study, followed by hydroxyvalerenic acid (2.72 +/- 0.63 microm/min, factor 0.07 related to diazepam) and acetoxyvalerenic acid (3.54 +/- 0.58 microm/min, factor 0.09 related to diazepam). To elucidate the contribution of the paracellular transport, studies were performed at different tightness status of the cell layers reflected by different transendothelial electrical resistance (TEER) values. Results showed an exponential correlation between transport and TEER for all three acids, whereas diazepam permeated TEER independently. In summary, it is hypothesized that the investigated compounds from Valeriana officinalis L. S. L. can probably only pass through the BBB by a still unknown transport system and not transcellularly by passive diffusion.     

建立体外共培养血脑屏障模型评价纳米粒的胞吞转运和毒性

目的建立大鼠脑毛细血管内皮细胞(BCECs)和星形胶质细胞共培养模型，评价纳米粒的跨血脑屏障(BBB)转运和对内皮细胞紧密连接的毒性。方法采用复乳/溶媒蒸发法制备载荧光探针6-香豆素的聚乙二醇-聚乳酸纳米粒。首先，分别从新生鼠大脑分离培养BCECs和星形胶质细胞并进行免疫组化鉴定。然后，将BCECs接种于细胞培养池微孔膜的上面，将星形胶质细胞接种于膜下面建立共培养模型。分别测定¹⁴C-蔗糖和纳米粒的渗透系数。结果载6-香豆素纳米粒的平均重均粒径为(102.4±6.8) nm，zeta电位为(-16.81±1.05) mV。BCECs的VIII因子表达呈阳性；星形胶质细胞的胶质原纤维酸性蛋白表达呈阳性。模型的跨内皮细胞电阻值为(313±23) Ω·cm²。扫描电镜和透射电镜观察发现，共培养模型中的BCECs形成紧密连接。纳米粒的质量浓度低于200 μg·mL^-1时，不影响¹⁴C-蔗糖渗透系数的改变，表明其不会影响BBB内皮细胞的紧密连接。10 μg·mL^-1载6-香豆素纳米粒的渗透系数为0.29×10^-3 cm·min^-1。结论该大鼠BBB模型与体内情况接近，适合用于评价纳米粒的脑内转运和毒性。     

Insulin and glucose induced changes in expression level of nucleoside transporters and adenosine transport in rat T lymphocytes

Adenosine is an endogenous agent exerting potent action on the immune system including regulation of lymphocyte functioning. Impaired T lymphocyte functioning is a common feature of diabetes. The aims of this study were to examine the effects of glucose and insulin on nucleoside transporters (NT) expression level and adenosine (Ado) transport in rat T lymphocytes cultured under the defined concentrations of glucose and insulin. Performed experiments revealed that rat T lymphocytes expressed the equilibrative nucleoside transporter type 1 and 2 (rENT1, rENT2) and concentrative nucleoside transporter type 2 (rCNT2). The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but were not affected by changes in extracellular glucose concentration. Exposition of T cells to 10nM insulin resulted in 73% increase in rENT2 mRNA and 50% decrease in the rCNT2 mRNA level. The level of rENT1 mRNA was sensitive to extracellular glucose concentration but not to insulin. The highest differences among cells cultured in high (20mM) and low (5mM) glucose were observed in equilibrative nitrobenzylthioinosine sensitive adenosine transport, which was lowered by 65% in cells cultured at high glucose. Alterations in adenosine transport were accompanied by changes in adenosine accumulation in the cell. These results indicate that adenosine transport in rat T lymphocytes is independently and differentially regulated by glucose and insulin by means of changes in the nucleoside transporters expression level. Altered adenosine transport has a great impact on its intracellular level. This suggests that under diabetic conditions adenosine action on T lymphocytes might be altered.     

Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug.

OBJECTIVE: The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood-brain barrier (BBB) during in vitro drug transport assays is essential for an accurate estimation of the permeability coefficient of a drug. METHODS: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug transport, sodium fluorescein (P(app,FLU), M(w) 376 Da) and FITC-labeled dextran (P(app,FD4), M(w) 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between P(app) and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). RESULTS: P(app,FLU) and P(app,FD4) in control experiments varied from 1.6 up to 17.6 (10(-6)cm/s) and 0.3 up to 7. 3 (10(-6)cm/s), respectively; while for individual filters P(app, FLU) was 4 times higher than P(app,FD4) (R(2)=0.97). As long as TEER remained above 131.Omega cm(2) for FLU or 122.Omega cm(2) for FD4 during the transport assay, P(app) remained independent from the basal permeability of the in vitro BBB. Below these TEER values, P(app) increased exponentially. This nonlinear relationship between basal BBB permeability and P(app) was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (P(FLU) and P(FD4)) were estimated to be 2.2+/-0.1 and 0.48+/-0.03 (10(-6)cm/s), respectively. In the experimentally challenged experiments, a reliable indication for P(FLU) and P(FD4) could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. CONCLUSIONS: The assessment of basal BBB permeability status during drug transport assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug transport assay are determined.     

An in vitro transport model for rapid screening and predicting the permeability of candidate compounds at blood–brain barrier

The aim of this study was to design and develop a simple in vitro blood–brain barrier (BBB) permeation model for elementarily and rapidly predicting the permeability of candidate compounds at BBB and further evaluating whether P-glycoprotein (P-gp) affects them across BBB. The model was mainly composed of cultured rat brain microvascular endothelial cells (rBMECs), glass contraption, and micropore membrane. First, we evaluated the model by morphological observation. Second, the restriction effects of paracellular transport were verified by measuring marker probes transport, and monitoring transendothelial electrical resistance (TEER) and leakage. Finally, protein expression and activity of P-gp were confirmed by carrying out Western blot analysis and polarized transport of rhodamine-123 (Rho123) in rBMECs. The rBMECs retained both endothelial cells and BBB features. The rBMECs model reproducibly attained approximately 130 Ω cm² on the steady-state TEER value, and displayed a barrier function to marker probes transport by decreasing the permeability. Protein band of 170 kDa manifested the existence of P-gp in the rBMECs, and the findings of cyclosporin A-sensitive decrease of Rho123 efflux confirmed the presence of P-gp activity. A simple, rapid, and convenient in vitro BBB permeation model was successfully established and applied to evaluate the BBB transport profiles of three natural flavonoids: quercetin, naringenin, and rutin.     

Interaction of nucleoside analogues with nucleoside transporters in rat brain endothelial cells

A number of nucleoside analogues, consisting of antiviral compounds and agents designed as adenosine A1 receptor agonists, were examined for nucleoside transporter affinity using an in vitro model of the blood-brain barrier (BBB), the rat brain endothelial cell line, RBE4. Structure-activity relationships (SAR) were also performed to identify the key structural requirements for transporter recognition and the suitability of these systems for carrier-mediated strategies to deliver therapeutics across the BBB. Adenosine receptor agonists did not show transport affinity for concentrative nucleoside carriers, but exhibited affinity for equilibrative systems (Ki=10.8-97.9 microM) within the range of Kms for natural substrates. However, none of the antiviral compounds tested in this study showed affinity for either class of nucleoside transporter. SAR studies suggest that the hydroxyl group located at the 3'-position of the ribose moiety is an essential requirement for transporter recognition. This may explain the inability of nucleoside derived anti-viral compounds to use these systems despite the significant structural homology with naturally occurring nucleosides. Sites have also been identified which accommodate structural additions with retention of carrier affinity, suggesting that compounds which fail to penetrate the BBB could be attached to these sites for carrier-mediated delivery using a prodrug strategy.     

Brain penetration of synthetic adenosine A1 receptor agonists in situ: role of the rENT1 nucleoside transporter and binding to blood constituents.

The blood-brain barrier (BBB) transport of synthetic A(1) receptor agonists was studied in an in situ brain perfusion model in the presence and absence of the selective nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI). For 8-methylamino-N(6)cyclopentyladenosine (MCPA), N(6)-cyclopentyladenosine (CPA), 2'deoxy-N(6)-cyclopentyladenosine (2'dCPA) and 5'deoxy-N(6)-cyclopentyl adenosine (5'dCPA) the brain uptake clearance was low with values of 0.0045+/-0.0012, 0.018+/-0.0020, 0.022+/-0.0028 and 0.12+/-0.054 ml min(-1)g(-1), respectively. In the presence of an average NBTI plasma concentration of 2.6+/-0.3 microg ml(-1) (NBTI dose: 3 mg kg(-1) i.v.) the values of the brain uptake clearance were 0.0062+/-0.0012, 0.013+/-0.0017, 0.014+/-0.0030 and 0.13+/-0.066 ml min(-1)g(-1), respectively and not significantly different from the values in the absence of NBTI. In a separate experiment the brain uptake of MCPA from phosphate buffered saline (PBS) and whole blood were compared. The brain uptake clearance from whole blood (0.0012+/-0.001 ml min(-1)g(-1)) was significantly lower than from PBS (0.0045+/-0.0012 ml min(-1)g(-1)). The results of these studies show that the rENT1 nucleoside transporter does not contribute significantly to the transport of synthetic A(1) receptor agonists across the BBB and that binding to blood constituents restricts the brain uptake.     

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell-surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A(1) receptor abundance. The regional co-localization of hENT1 and A(1) receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A(1) receptors in human brain.     

Nucleoside transporter subtype expression and function in rat skeletal muscle microvascular endothelial cells

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.     

Mechanisms of the Penetration of Blood-Borne Substances into the Brain

The blood-brain barrier (BBB) impedes the influx of intravascular compounds from the blood to the brain. Few blood-borne macromolecules are transferred into the brain because vesicular transcytosis in the endothelial cells is considerably limited and the tight junction is located between the endothelial cells. At the first line of the BBB, the endothelial glycocalyx which is a negatively charged, surface coat of proteoglycans, and adsorbed plasma proteins, contributes to the vasculoprotective effects of the vessels wall and are involved in maintaining vascular permeability. In the endothelial cytoplasm of cerebral capillaries, there is an asymmetrical array of metabolic enzymes such as alkaline phosphatase, acid phosphatase, 5’-nucleotidase, adenosine triphosphatase, and nucleoside diphosphatase and these enzymes contribute to inactivation of substrates. In addition, there are several types of influx or efflux transporters at the BBB, such as P-glycoprotein (P-gp), multidrug resistance associated protein, breast cancer resistance protein, organic anion transporters, organic cation transporters, organic cation transporter novel type transporters, and monocarboxylic acid transporters. P-gp, energy-dependent efflux transporter protein, is instrumental to the barrier function. Several findings recently reported indicate that endothelial P-gp contributes to efflux of undesirable substances such as β-amyloid protein from the brain or periarterial interstitial fluid, while P-gp likely plays a crucial role in the genesis of multiple vascular abnormalities that accompany hypertension. In this review, influx and efflux mechanisms of drugs at the BBB are also reviewed and how medicines pass the BBB to reach the brain parenchyma is discussed.Key Words: Blood-brain barrier, P-glycoprotein, tight junction.